Membrane proteins make up over 30% of the human proteome and are critical in biological functions, including transport and the transduction of cellular information. Membrane proteins also feature in the multistep pathways that lead to glycoproteins. For example, the human pathway for protein N-glycosylation, occurs exclusively at cellular membranes and is part of the essential process that ultimately affords all cell- surface and secreted N-linked glycoproteins. Likewise, bacterial, glycoproteins are generated through stepwise, membrane-associated glycan assembly pathways, which culminate in the biosynthesis of important virulence-associated glycoproteins. Despite the widespread importance of multistep membrane pathways in human health and disease and the extensive knowledge on the enzymes that make up these pathways, our understanding of how the enzymes function together and in an ordered sequence are greatly challenged by technical issues associated with the amphiphilic nature of membranes and the properties of associated membrane-bound proteins. Functional studies on membrane proteins are often simplified by extraction into detergent micelles. However, this treatment is highly perturbing and under these conditions, all but the most stable multiprotein complexes will dissociate and the cryptic information that is programmed in the native membrane will be lost. Therefore, a major current challenge is to develop strategies for understanding how proteins are recruited into functional complexes at cellular membranes. This challenge demands the application of synergistic in vivo and in vitro experimental approaches. The proposed research will investigate the membrane-associated protein N-glycosylation pathway of the Gram-negative enteropathogen Campylobacter jejuni, which shares the logic of the more complex mammalian pathway. The research has three aims. In Aim 1 we will define the membrane protein interactome for bacterial N-glycosylation using styrene maleic acid lipoparticles (SMALP). SMALP will enable definition of a complete molecular description of the local membrane environment around target membrane proteins in vivo. In Aim 2 we will leverage the interactome information for in vitro studies in lipid bilayer Nanodiscs (NDs), which provide a native-like model membrane of defined composition, and enable in vitro experimental approaches to understand the rules defining the membrane protein interactions and coordinated function. In Aim 3 we will implement crosslinking studies to address a key question concerning the timing of glycan transfer to protein. Ultimately, the studies will deliver detailed information regarding the membrane environment and protein interaction network that supports efficient N-glycosylation in a representative bacterial pathway. If successful, the research will provide insight into the molecular logic underpinning the processes that lead to glycoprotein biosynthesis in all living systems and will inform on multidisciplinary approaches for investigating other physiologically significant multienzyme processes that occur at membrane interfaces.